This invention relates generally to compositions of and methods for obtaining peroxisome proliferator-activated receptorxcex3 polypeptides. The invention relates as well to the DNA sequences encoding these receptor polypeptides, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant peroxisome proliferator-activated receptorxcex3 polypeptides. The invention includes, as well, methods for using the isolated, recombinant receptor polypeptides in assays designed to select and peroxisome proliferator-activated receptor-xcex3 polypeptides for use in diagnostic, drug design and therapeutic applications.
The steroid hormone (or nuclear) receptor superfamily has many is members that play important roles in regulating cancer cells, including, but not limited to, the estrogen receptor (Bettuzi, 1991), the retinoic acid receptors (Menger, 1988), and v-erb A (Sharif, 1991). A number of less well characterized members of this family have been isolated and identified based on their homology to the better known nuclear receptors. (Laudet, 1992). Many show ligand-dependent activation patterns, but the identity of natural ligands that control these transcription factors is still unknown, or under investigation. Members of the steroid hormone receptor family whose ligands have not yet been identified are referred to as xe2x80x9corphanxe2x80x9d receptors. The full importance of these xe2x80x9corphanxe2x80x9d receptors is predicted by their presence early in development and by the fact that many (including PPAR, COUP, ROR) have been shown to interact with the known members of the superfamily (Beck, 1992; O""Malley, 1992; Tsukiyama, 1992). Many of these receptors are undoubtedly important in regulating cell growth and response to the environment, and may well have regulatory roles in cancer cells, like their currently well-studied counterparts.
The present invention provides a novel xe2x80x9corphanxe2x80x9d receptor polypeptide, the human Peroxisome Proliferator Receptor xcex3 (hPPAR-xcex3). It is a member of a group of orphan receptors found to be activated (but not bound) by arachidonic acid, fatty acids, clofibrate, and other agents that induce the proliferation of peroxisomes in rodents. (Blaauboer, 1990).
Peroxisome proliferators are a diverse group of chemicals which include hypolipidemic drugs, herbicides, leukotriene antagonists, and plasticizers. Two major categories of peroxisome proliferator chemicals play a significant role in society today. The first, the fibrate class of hypolipidemic drugs, has been found to be effective at reducing the levels of triglycerides and cholesterol in humans suffering from hyperlipidemia, a major risk factor for heart disease (Berioli, 1990). The second category relates to phthalate ester plasticizers used in the production of highly versatile flexible vinyl plastics (Reddy, 1983).
Peroxisome proliferators seem to affect most mammalian species that have been tested. They induce hepatomegaly resulting from liver hyperplasia and an increase in the size and number of peroxisomes. (Reddy, 1983.) Nevertheless, on the basis of hypolipidemic drug dose required to produce recognizable peroxisome proliferation, mice and rats are considered to be highly responsive to these agents, developing hepatocellular carcinoma following long-term drug administration; hamsters have intermediate responses; and guinea pigs, marmosets and other nonhuman primates are weakly responsive (Eacho, 1986).
Peroxisome proliferators are termed non-genotoxic carcinogens as they fail to cause DNA damage directly (Warren, 1980). The increase in peroxisomal fatty acid xcex2-oxidation seen in response to peroxisome proliferators results in a greater production of hydrogen peroxide. It has been proposed that this results in oxidative stress leading to DNA damage and possible tumor initiation. (Reddy, 1983; Kasai, 1989). Alternatively, or in addition, peroxisome proliferators may act as liver-tumor promoters. (Marsman, 1988; Cattley, 1989).
The nuclear receptor subfamily to which the receptor polypeptide of the present invention belongs has been shown to regulate the transcription of several key enzymes in fatty acid metabolism, including Acyl Co A oxidase (Tugwood, 1992), and CYP4A6, a cytochrome P-450 omega hydroxylase, potentially having profound effects upon host responses via leukotriene and prostaglandin catabolism, fatty acid xcex2 oxidation, and superoxide production. Host response is a key aspect to the pathology of all diseases, including cancer, infection, and autoimmune disorders.
Recent studies demonstrate the PPARs can heterodimerize with the 9-cis retinoic acid receptor (RXR) and synergistically induce gene expression (Kliewer, 1992). These studies demonstrate a potential link between the retinoid responsive pathways and the lipid/arachidonic acid metabolic pathways. Preservation of the hydrophobic heptad repeats in the dimerization domain (see discussion below) indicates that PPARs can heterodimerize with other members of the thyroid/retinoid subbranch of the superfamily containing these repeats. There is also some conservation of the xe2x80x9cconserved activating motifxe2x80x9d (Daneilian et al., 1992) in the carboxy terminus.
PPARs are orphan receptors described in the murine (Issemann, 1990), rat (Gottlicher, 1992), human (Sher, 1993) and xenopus systems (Dreyer, 1992) as the peroxisome proliferator activated receptors xcex1, xcex2, and xcex3. A fourth member (h NUC 1) with some interesting unique sequence characteristics was isolated from human osteosarcoma cells (Schmidt, 1992). These receptors in other species are known to be activated by arachidonic acid at 150 xcexcM levels, long chain fatty acids such as oleic and petroselenic acid, clofibrate, and other peroxisome proliferating agents (Gottlicher, 1992; Isseman, 1990). Arachidonic acid can trigger many different responses in cells such as neutrophils, but PPAR activation by long chain fatty acids provides evidence that arachidonic acid metabolites are candidate ligands or activators. In addition, murine PPAR-xcex1 has been shown to transactivate as a heterodimer with the human Retinoid X Receptor alpha (hRXR-xcex1) both on retinoid receptor targets as well as on known PPAR target promoter sequences, potentially linking these pathways of gene regulation (Kliewer, 1992).
PPARs may play a role in proliferative and differentiation aspects of cancer, because they have also been shown to be developmentally active in vertebrates (xenopus), and are present in oocytes, fertilized eggs, blastulae, gastrulae, neurulae, and early tadpoles (Dreyer, 1992). It should be noted that many other members of the nuclear receptor superfamily appear to have two sets of regulatory functions. The first occurs during embryogenesis and development, including axis and pattern formation (Blumberg, 1992), and the second occurs in the adult, where the receptors modulate transcriptional activities in response to neighboring cells, other tissues, and aspects of the environment, particularly on regenerating tissues (Elder, 1991; Howell, 1990).
Members of the superfamily of nuclear hormone receptors have also been shown to directly connect the cellular transcriptional response to extracellular signals such as retinoids and steroids, as well as fatty acid and arachidonic acid metabolites, as already mentioned (Aronica, 1991). Studies of these receptors in hematopoiesis have primarily focused upon the retinoic acid receptor alpha (RAR-xcex1) and v-erb A genes because of their important effects on hematopoietic cell differentiation.
The retinoic acid receptor xcex1 gene on chromosome 17 is translocated to chromosome 15 in virtually all cases of human acute promyelocytic leukemia (APL) (Rowley, 1988), where it is fused with PML gene. This generates two fusion proteins, containing RAR-xcex1 and PML sequences, which have different transcriptional activating properties than the wild type proteins. Treatment of APL patients with the ligand, all trans retinoic acid, consistently results in the achievement of a complete remission in this disease (Menger, 1988).
V-erb A is an aberrant version of a thyroid hormone receptor that can block erythroid differentiation and induce malignant transformation, an ability that is correlated with the repression of retinoic acid receptor function. Both retinoic acid receptors and v-erb A elicit significant effects upon the hematopoietic system by interacting with other transcripti on factors, such as fos and jun (Schule, 1991), and with other related nuclear receptors leading to heterodimer formation (Debois, 1991). The retinoic acid receptors, (RARs) can heterodimerize with retinoid x receptors (RXRs) in vitro, which increases their affinity for the RAR response element. RXRs can also heterodimerize with a number of different members of the superfamily in vitro, including the PPARs (Kliewer, 1992).
Target genes identified as transcriptionally activated by PPARs include acyl co-A oxidase, the key enzyme regulating the fatty acid B oxidation pathway (Tugwell, 1992), and CYP4A6, a cytochrome P450 fatty acid xcfx89 hydroxylase, a key enzyme catalyzing the xcfx89 hydroxylation of arachidonic, lauric and palmitic acids (Muerhoff, 1992). The sequence in the rat acyl-Co A oxidase promoter bound by PPARs is xe2x80x9cacgTGACCTtTGTCCTggtxe2x80x9d (SEQ ID NO:6) (Tugwell, 992), which contains a direct repeat, separated by one nucleotide, (xe2x80x9cDR-1xe2x80x9d motif) (Umesono, 1991) of the canonical consensus half site xe2x80x9cTGACCTxe2x80x9d (SEQ ID NO:7) to which all members of the thyroid-retinoid branch of the nuclear receptor superfamily can bind (Laudet 1992). The target sequences in CYP4A6 appear as imperfect DR-1 motifs and more complex arrangements of imperfect half sites. Candidate gene promoters must be tested for PPAR activation, but genes such as CD18, the leukocyte integrin xcex2 subunit, which has multiple combinations of imperfect half-sites and is retinoic acid inducible (Agura, 1992), are possibly PPAR responsive. Catalase, hydroxyacid oxidase, and uricase are down regulated by peroxisome proliferators in the rat, under the same conditions that up regulate acyl-coA oxidase. The effects of PPAR upon myeloperoxidase, chloroacetate esterase or catalase in myeloid cells have not been examined. Further characterization of these two forms of the PPAR-xcex3 mRNA will be extremely useful for understanding their functions in hematopoietic cells as well as other organ systems.
In one aspect, the present invention provides an isolated and purified polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide. In a preferred embodiment, a polynucleotide of the present invention is a DNA molecule. More preferably, a polynucleotide of the present invention encodes a polypeptide that is a peroxisome proliferator-activated receptor-xcex3. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequence of SEQ ID NO:1.
In an alternative aspect, the present invention contemplates an isolated and purified polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide, said polynucleotide preparable by a process comprising the steps of (a) constructing a library of cDNA clones from a cell that expresses said polypeptide; (b) screening the library with a radio-labelled oligonucleotide probe; (c) identifying a clone that hybridizes to the probe; and (d) isolating the hybridized clone from the library of unhybridized clones. Preferably, the present invention provides an isolated and purified polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide prepared by a process comprising the steps of (a) constructing a library of cDNA clones from a cell that expresses said polypeptide; (b) screening the library with a radio-labelled oligonucleotide probe; (c) identifying a clone that hybridizes to the probe; and (d) isolating the hybridized clone from the library of unhybridized clones. More preferably, the polypeptide encoded has the amino acid residue sequence of SEQ ID NO: 2. Still more preferably, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1.
Yet another aspect of the present invention contemplates an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 10 contiguous bases of SEQ ID NO: 1, wherein the polynucleotide hybridizes to a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide. Preferably, the isolated and purified polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of SEQ ID NO: 1. For example, the polynucleotide of the present invention can comprise a segment of nucleotide bases identical or complementary to 40 or 55 contiguous bases of the disclosed nucleotide sequences.
In still another embodiment of the present invention, there is provided an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 10 contiguous bases of SEQ ID NO: 1. The polynucleotide of the invention hybridizes to SEQ ID NO: 1, or a complement of SEQ ID NO: 1. Preferably, the isolated and purified polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of SEQ ID NO: 1. For example, the polynucleotide of the invention can comprise a segment of bases identical or complementary to 40 or 55 contiguous bases of SEQ ID NO: 1.
In another embodiment, the present invention contemplates an isolated and purified peroxisome proliferator-activated receptor polypeptide. Preferably, a peroxisome proliferator-activated receptor polypeptide of the invention is a recombinant polypeptide. More preferably, a peroxisome proliferator-activated receptor polypeptide of the present invention is a peroxisome proliferator-activated receptor-xcex3 polypeptide. Even more preferably, a peroxisome proliferator-activated receptor polypeptide of the present invention comprises the amino acid residue sequence of SEQ ID NO:2.
In an alternative embodiment, the present invention provides an expression vector comprising a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide. Preferably, an expression vector of the present invention comprises a polynucleotide that encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2. More preferably, an expression vector of the present invention comprises a polynucleotide comprising the nucleotide base sequence of SEQ ID NO:1. Even more preferably, an expression vector of the invention comprises a polynucleotide operatively linked to an enhancer-promoter. More preferably still, an expression vector of the invention comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector of the present invention comprises a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter, and the expression vector further comprises a polyadenylation signal that is positioned 3xe2x80x2 of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.
In yet another embodiment, the present invention provides a recombinant host cell transfected with a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide. Preferably, a recombinant host cell of the present invention is transfected with the polynucleotide of SEQ ID NO:1. Even more preferably, a host cell of the invention is a eukaryotic host cell. Still more preferably, a recombinant host cell of the present invention is a yeast cell. Alternatively, a recombinant host cell of the invention is a COS-1 cell. In another aspect, a recombinant host cell of the present invention is a prokaryotic host cell.
In yet another embodiment, the present invention contemplates a process of preparing a peroxisome proliferator-activated receptor polypeptide comprising transfecting a cell with polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide to produce a transformed host cell; and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide. Preferably, the transformed host cell is a eukaryotic cell. More preferably still, the eukaryotic cell is a COS-1 cell. Alternatively, the host cell is a prokaryotic cell. More preferably, the prokaryotic cell is a bacterial cell of the DH5xcex1 strain of Escherichia coli. Even more preferably, a polynucleotide transfected into the transformed cell comprises the nucleotide base sequence of SEQ ID NO:1.
In still another embodiment, the present invention provides an antibody immunoreactive with a peroxisome proliferator-activated receptor polypeptide. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the antibody is immunoreactive with a peroxisome proliferator-activated receptor polypeptide that comprises the amino acid residue sequence of SEQ ID NO:2.
In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with a peroxisome proliferator-activated receptor polypeptide comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing the antibody to the polypeptide. Preferably, the host cell is transfected with the polynucleotide of SEQ ID NO:1. Even more preferably, the present invention provides an antibody prepared according to the process described above.
Alternatively, the present invention provides a process of detecting a peroxisome proliferator-activated receptor polypeptide, wherein the process comprises immunoreacting the polypeptide with an antibody prepared according to the process described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
In yet another embodiment, the present invention contemplates a process of detecting a messenger RNA transcript that encodes a peroxisome proliferator-activated receptor polypeptide, wherein the process comprises (a) hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes the peroxisome proliferator-activated receptor polypeptide to form a duplex; and (b) detecting the duplex. Alternatively, the present invention provides a process of detecting a DNA molecule that encodes a peroxisome proliferator-activated receptor polypeptide, wherein the process comprises (a) hybridizing DNA molecules with a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide to form a duplex; and (b) detecting the duplex.
Alternatively, the present invention contemplates a pharmaceutical composition comprising a peroxisome proliferator-activated receptor polypeptide and a physiologically acceptable carrier. Preferably, the present invention provides a pharmaceutical composition comprising a receptor polypeptide that comprises the amino acid residue sequence of SEQ ID NO:2. In another embodiment, the present invention provides a pharmaceutical composition comprising a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide, and a physiologically acceptable carrier. Preferably, that pharmaceutical composition comprises a polynucleotide encoding a receptor polypeptide comprising the amino acid residue sequence of SEQ ID NO:2. Even more preferably, the pharmaceutical composition comprises a polynucleotide that comprises the nucleotide sequence of SEQ ID NO:1.
In another aspect, the present invention contemplates a diagnostic assay kit for detecting the presence of a peroxisome proliferator-activated receptor polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with a peroxisome proliferator-activated receptor polypeptide, with the first antibody present in an amount sufficient to perform at least one assay. Preferably, an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody. More preferably, the antibodies used in an assay kit of the present invention are monoclonal antibodies. Even more preferably, the first antibody is affixed to a solid support. More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label or an enzyme.
In an alternative aspect, the present invention provides a diagnostic assay kit for detecting the presence, in biological samples, of a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of SEQ ID NO:1.
In another embodiment, the present invention contemplates a diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a peroxisome proliferator-activated receptor polypeptide, the kit comprising a first container containing a peroxisome proliferator-activated receptor polypeptide that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
In yet another aspect, the present invention contemplates a process of screening substances for their ability to interact with a peroxisome proliferator-activated receptor polypeptide comprising the steps of providing a peroxisome proliferator-activated receptor polypeptide, and testing the ability of selected substances to interact with the peroxisome proliferator-activated receptor polypeptide.
In a preferred embodiment, providing a peroxisome proliferator-activated receptor polypeptide is transfecting a host cell with a polynucleotide that encodes a peroxisome proliferator-activated receptor polypeptide to form a transformed cell and maintaining the transformed cell under biological conditions sufficient for expression of the peroxisome proliferator-activated receptor polypeptide.